Pernicious anaemia refers to a form of anaemia, which results from failure of the body to absorb adequately absorb vitamin B-12. Vitamin B-12, being an important nutrient in the body is absorbed in the small intestine from foods rich in vitamin B-12. Lahner and Annibale (2009) explain that vitamin B-12 binds itself to intrinsic factor, a parietal-cells-secreted protein, forming a complex, which is readily absorbed by the small intestine.
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Irvine (1965) submits that when the aforementioned intrinsic factor is absent from the body, perhaps because of autoimmunity of genetic issues, vitamin B-12 is reduced in the victim’s body, leading to pernicious anaemia.
Further, victims of autoimmune disorders are more exposed to pernicious anaemia because their antibodies attack parietal cells, leading to abnormal secretion of the intrinsic factor, which in turn leads to poor vitamin B-12 absorption and consequently, pernicious anaemia (Toh, Van Driel & Gleeson, 1997). Therefore, the pathophysiology and pathogenesis of pernicious anaemia comes from the autoimmune disorder.
The pathophysiology and pathogenesis of pernicious anaemia show that occurrence of autoimmune disorder affects secretion of the intrinsic factor leading to mal-absorption of vitamin B-12.
Autoantibodies attach themselves to gastric H+/K+–ATPase that is a pump composed of a 100 a glycoprotein β-subunit (60-90 kDa) and a kDa catalytic α-subunit, and consequently prevent the proper functioning of parietal cells and intrinsic factor secretion (Toh et al., 1997).
This report is an investigative analysis of the pathophysiology and pathogenesis of pernicious anaemia. It analyses the stomach’s morphology in relation to pernicious anaemia, diagnoses pernicious anaemia in sample stomachs through western blot technique, and seeks to detect antibodies in sample serums through immune-histochemistry staining.
The objective of this report is to detect antibodies of the proton pump of the stomach by applying immune-histochemistry and western blot techniques on serum samples. To set a good background for the study, the report first analyses the critical components of a healthy stomach.
Material and Methods
Procedure of Examining Morphology of the Stomach
Stomach samples were placed in slides to prepare them for examination, and then stored in a safety cabinet. The slides were incubated in xylene for two minutes and further incubated in ethanol for another two minutes. Rinsing was done on the slides for 30 seconds after the slides were incubated in haematoxylin. Staining and washing was done, followed by soaking in 1% acid alcohol and rinsing.
The stained slides were placed for 30 seconds in Scott’s tap to incubate, and thereafter, rinsed before staining them with eosin for a period of 4 minutes. The slides were subsequently placed for 30 seconds in 90% and 100% ethanol to fix them.
After the fixation, the slides were put on ethanol for 2 minutes and dried in the air immediately. A drop of DPX medium was applied on fixed slide, preparing it for mounting, covered with cover slip, and a magnification of x400 used to view it. Diagrams focussing on the gastric gland and stomach wall were sketched using a pencil on an A4 paper.
Procedure for Preparing SDS-PAGE and Western Blot
For the stomach specimen, a protein sample of 48µL was added into a microfuge with 12µL of SDS-sample. Molecular weight markers for the assessment of the molecular weights were prepared in advance. To sediment the molecular markers and protein sample, the solutions were spun in a centrifuge for a few minutes.
Next, lane 1 of the prepared SDS-PAGE in electrophoresis apparatus was loaded with 10µL of the molecular weight markers. Just like molecular weight markers, subsequent wells of the SDS-PAGE were loaded with 25µL of the protein sample. Then the electrophoresis apparatus was started on a power of 200 volts and left for 45 minutes.
After electrophoresis was over, the apparatus was switched off and dismantled, and a demonstrator was used to apply gel on a clean plate. Then the gel was placed on the iBlot device with the aim of assessing the separation of proteins according to their weights. 10uL of distilled water was used in the placement of one gel in iBlot device, which processed it for 7 minutes, and another gel was put in nitrocellulose film.
The nitrocellulose membrane was stained using 50mL of 0.1% Ponceau solution and leaving it for 1 minute, which ensured that protein transformation had occurred. To detect sera of patients, the nitrocellulose membrane was labelled.
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Lanes were cut using a pair of scissors and subsequently rinsed using NaOH with an aim of removing stains. The membrane was washed in 50mL of TBS and stored for the subsequent practical, which would include 10mL of Tris-buffer at 40o C.
Deionised water was used to clean membranes after they were retrieved from storage. The wash included the following three steps: blocking step involving 5mL of 5% skimmed milk blocking solution; incubation of primary antibody using 2.5mL of patient serum sample or the positive and the negative control; and secondary antibody incubation using 5mL of labelled antibody.
The iBlot was put to use again in performing the aforementioned three steps. Consequently, membrane strips were removed from the device and washed with TBS 3 times for a period of five minutes. Several steps were subsequently performed before imaging using the Chemidoc. The results of the test were then uploaded to CloudDeakin.
Procedure of Preparing Immuno-Peroxidase of Mouse Stomach
As some steps of slides preparation was done beforehand, Incubation was done for 100µL of serum for 20 minutes because some slide preparation steps had already been completed. Slides were washed using PBS horseradish peroxide, also known as anti-human Ig HRPO, was used to conjugate 50µL of antibodies by incubating it for 45 minutes.
Slides were then washed in PBS and water was used to rinse them before 100µL of DAB was added, subsequently incubating them for 10 minutes.
Before the slides were soaked in haematoxylin for 3 seconds, they were cleaned using PBS. After the cleaning, they were rinsed with water and incubated in ethanol for an approximate 2 minutes. DPX was applied on slides and they were viewed with a microscope magnification of ×400.
Discussion: Morphology of the Stomach
In order to understand the morphology of the stomach well, the mucosal and sub-mucosal layers of the mouse’s stomach are stained with eosin and haematoxylin as depicted in figure 1. Figure 2 shows gastric gland stained with the same, which together with the other parts are key determinants of the mouse’s stomach morphology.
As Irvine (1965) notes, the major feature of pernicious anaemia is the atrophy of mucosal cells for example parietal cells. The latter are responsible for the secretion of gastric acid and intrinsic factor which aid in vitamin B-12 absorption in the end of the small intestine. “Essentially, parietal cells have the H+/K+-ATPase pump, which consists of beta and alpha subunits” (Rhoades & Bell 2008).
This is well illustrated in figure 2 whereby the parietal cells are pink stained while the other main cells (chief cells) are dark-pink stained. The chief cells play a significant role of pepsinogen secretion, which catabolizes proteins (Toh et al. 1997). In that case, the physiology of parietal cells and protein catabolism is dependent on the stomach morphology.
Discussion: Western Blotting
The experiment proved that a western blot technique is effective in determining whether patients are infected with pernicious anaemia. Results from the study were presented on CloudDeakin. It is however important to mention that the western blot technique did not achieve the desired results in all experiments because its results were spurious.
Some membranes in the study lacked bands, while some had patchy or uneven spots as shown in figure 3. Defects in antigens, buffers, or antibodies resulted in interference of definite bands in the membranes. Additionally, use of inappropriate antibodies, either primary or secondary, also resulted in vague bands or no bands at all.
Likewise, the use of low concentrations of the antibody also results in invisible bands and lack of antigen or low concentrations of the same leads to an invisible signal. In this regard, it is necessary to use different antigens in order to determine whether invisibility is because of primary and/or secondary antibodies or antigens.
The visibility of bands can also be affected by procedures like membrane washing. This is because prolonged washing is likely to diminish signal appearance. Contamination of buffers may also lead to invisible bands, hence the need to preserve their purity during the study (Lahner & Annibale, 2009). The western blot procedure should be undertaken in such a way that buffers like TBST, ECL and PBS are not contaminated.
The procedure’s transfer of protein component is perhaps the most sensitive because it may be interfered by air bubbles and partial transfer can occur (Andres & Serraj, 2012).
When such improper protein transfer occurs, then the blot comes out with uneven or patchy spots. In the event of trapped air bubbles within the membranes and gel, the output in film will be dark. Therefore, to prevent bubble formation, one should evenly distribute particles by incubating using a shaker.
The results presented in figure 4 were prepared by the technical staff. The H+/K+-ATPase antigen is responsible for triggering an autoimmune disorder (Lahner & Annibale, 2009), which interferes with the proper functioning of parietal cells and intrinsic-factor secretion (Lahner & Annibale, 2009).
The parietal cells’ autoantibodies act against H+/K+-ATPase, thereby affecting the function of parietal cells, and consequently the occurrence of pernicious anaemia (Andres & Serraj, 2012).
Structurally, the autoantigen measures an approximate 160 to 190 kDa, with the specific measurement being determined by its constituent protein subunits. The autoantigen is made up of a glycoprotein β-subunit (60-90 kDa) and a 100 kDa catalytic α-subunit (Toh et al., 1997). The subunits are the ones that determine the nature of the disorder affecting parietal cells and intrinsic factor secretion.
The western blot analysis revealed a fragment of approximately 80 kDa. The fragment is a glycosylated β- subunit of the H+/K+-ATPase pump because the fragment detected corresponds a 80 kDa molecular marker. In the event of pernicious anaemia, H+/K+-ATPase is the autoantigen that is solely recognizable by parietal-cells antibodies. Therefore, it is vital in diagnosing pernicious anaemia using western blot (Toh et al., 1997).
Evidently, patient 1’s stained strip in figure 4, indicates a positive diagnosis. The size of the autoantibody-bound subunit is approximately 80 kDa that is a glycosylated β-subunit of the H+/K+-ATPase pump. Therefore, the findings from the western blot procedure confirm that the patient is positive, and that the autoantigen subunit is glycosylated β- subunit of the H+/K+-ATPase pump.
ELISA, which is an acronym for Enzyme linked immunosorbent assay, can also be utilized in detecting anti-proton pump antibodies that prove presence of pernicious anaemia in patients. H+/K+-ATPase is the main autoantigen, which is present in pernicious-anaemia patient sera because it induces autoimmune disorder that interferes with parietal cells and thereby inhibits secretion of the intrinsic factor (Lahner & Annibale, 2009).
When ELISA is used, the autoantigen on the micro titre plate is immobilized, and the serum sample that contains anti-proton pump antibodies is combined and incubated with the aforementioned antigen. Serum samples normally have antibodies that are specific to proton pump antigens.
This implies that the antibodies bind to the proton pump α and β subunits. Sugiu et. al. explain that proteins and excess antibodies in serum are cleaned and the secondary antibody that is linked to the enzyme, and which is primary-antibody specific, is also added to the micro titre plate. A change in colouration is achieved by the use of the chromogenic substrate, indicating proton-pump antibodies presence.
In order to be able to determine the amount of proton pump antibodies in sera by the use of ELISA, the assessment of electrical signal, intensity of colour and fluorescence is necessary.
To measure colour intensity in absorbance of luminance, it is a spectrophotometer can be used (Sugiu et al., 2006). This accurately determines the level of proton pump antibodies in the sera. Therefore, determination of fluorescence or colour intensity is an important component of qualifying proton pump antibodies.
The anti-human IG-HRP conjugate of the sheep is a secondary antibody, which links with the H+/K+ ATPase antigen indirectly because it uses the primary antibody to link to the antigen. Anti-human Ig-HRP is actually an antibody specific to the human Ig production of the antibody, and it results from the immunological response of a sheep to human Ig (Chevrier, Chateauneuf, Guerin & Lemieux, 2004).
The anti-human Ig is actually a secondary antibody and thus, immune-histochemistry and in western blot, horseradish is used to conjugate it. Horseradish is an enzyme, which catalyses chromogenic substrates.
Discussion: Immuno-peroxidase staining of mouse stomach section
In this section of the report, emphasis is put on the stomach’s immune-histology to determine whether the patient mouse tests positive or negative for pernicious anaemia. In case of positive results, the antibodies are seen attached on the proton pumps of the parietal cells (Irvine 1965). During this experiment, unknown patients were applied anti-human 1g to the human 1g, which were labelled P1 and P2.
From the experiment results, it was depicted that there were no brown stains thus concluding that there was no antibody-attachment on the proton pumps of the parietal cell.
The conclusion was that patient 2 had tested negative for pernicious anaemia. On the other hand, patient 1 tested positive for pernicious anaemia because there were brown stains, which indicated antibody-antigen attachment on the proton pumps of the parietal cells.
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